The cytotoxic effect of the vitamin B12 inhibitor cyanocobalamin [c-lactam], and a review of other vitamin B12 antagonists

The vitamin B12 antagonist cyanocobalamin [c-lactam] was cytotoxic to cultured human leukemia cells, grown in methylfolate, homocysteine, and vitamin B12, but not in the presence of methionine. Small concentrations of methionine were effective in restoring the growth rate in a dose-dependent fashion, confirming methionine deficiency as the cytotoxic principle. Cyanocobalamin [c-lactam] prevented utilization of the methyl group of methylfolate, but no evidence of folate deficiency developed in long-term culture. High concentrations of non-methylated folate were unable to reverse the cytotoxicity, excluding a methylfolate 'trap' as the cause. Low concentrations of serine in the medium induced transient biochemical megaloblastosis. Cyanocobalamin [c-lactam] caused this to occur earlier, and persist. In high concentrations of serine, the inhibitor caused only transient changes in deoxyuridine suppression. Homocysteine cannot be remethylated without vitamin B12, and condensation with serine is the only other excretory pathway for this toxic amino acid. We hypothesize that impaired DNA synthesis in vitamin B12 deficiency is the result of diverting serine away from thymidylate synthesis, into homocysteine metabolism.     

Serum vitamin B12 levels in the aged

Stapedius-reflex measurement and the glycerol-test in combination with pure tone audiogram and speech-audiogram are reliable methods for an exact diagnosis of Menière's disease. For differential diagnosis one should know the so called "tensor tympani syndrome" which is discussed in this paper.     

The absorption of vitamin B12 after subtotal pancreatectomy in the rat

Subtotal pancreatectomy in the rat was performed by a new method. Except for a small remnant along the splenal hilus, the gland was completely removed. Exocrine pancreatic insufficiency was confirmed by fat and nitrogen malabsorption. A moderate diabetes mellitus was seen. Insulin substitution or diet restriction was unnecessary. The absorption of vitamin B12 after oral administration of 10 ng 57CoB12 was 5.0 +/- 0.2 (ng 57CoB12 +/- S.E.M.) in unoperated rats and 5.1 +/- 0.1 in pancreatectomized rats (p greater than 0.5). Pancreatic extract (PE) (100 mg) increased the absorption of 57CoB12 in unoperated rats (p less than 0.01), but not in pancreatectomized rats (p greater than 0.2). PE (300 mg) reduced the absorption in unoperated rats (p less than 0.01). The results indicate that an optimal amount of pancreatic secretion may stimulate the absorption of vitamin B12.     

Intestinal absorption of vitamin B12 in patients with chronic pancreatic insufficiency and the effect of human duodenal juice on the intestinal uptake of vitamin B12

The mean absorption of vitamin B12 (Schilling test) was 13.1 +/- 1.0 (% +/- S.E.M.) in 21 patients with chronic pancreatic insufficiency and 17.6 +/- 1.4 in 13 control patients (p less than 0.01). There was no correlation between pancreatic bicarbonate production after secretion stimulation and vitamin B12 absorption in the patient group (r = 0.117). Human duodenal juice reduced the uptake of 57CoB12-rat intrinsic factor (IF) by perfused rat small intestinal segments in vivo (p less than 0.01) as well as the uptake of 57CoB12-human IF by purified guinea-pig intestinal brush borders in vitro (p less than 0.01). The results confirm reduced absorption of vitamin B12 in chronic pancreatic insufficiency, but the mechanism remains uncertain.     

Radioisotopic assay of vitamin B12 in tissues

The present study delineates the application of the radioisotopic competitive-inhibition assay for the measurement of vitamin B12 in tissues. The extraction of endogenous B12 from tissues was shown to be simple and complete. Proportional dilution studies suggest that tissue factors do not interfere with the assay. Although some variability exists when multiple areas of an organ are sampled, the differences between B12 levels in tissues obtained from normal individuals and B12-deprived individuals are so wide that individual intra-organ variability is trivial, As this tissue B12 assay is similar to the widely utilized radioisotopic assays for serum B12, it is applicable for routine use.     

Physician's management of suspected vitamin B12 deficiency

A retrospective study was undertaken to audit physician's management of patients with a low serum level of vitamin B12 who were admitted to a university-affiliated teaching hospital during 1 year. Among the 34 patients 13 were proved to have pernicious anemia or vitamin B12 malabsorption, but for 12 of them there were unnecessary delays (several days or weeks) before initiation of investigation and therapy. An additional six patients, who had low serum levels of vitamin B12 and macrocytosis, most likely had true vitamin B12 deficiency, but proper investigation was not done and they did not receive any vitamin B12 or folic acid therapy. In another nine cases unexplained low serum levels of vitamin B12 were not properly investigated, and the patients either did not receive any vitamin B12 therapy or received it without proper documentation of a deficiency. Suggestions for facilitating early detection, investigation and treatment of megaloblastic anemia or vitamin B12 deficiency are given.     

The effect of vitamin B12 deficiency on older veterans and its relationship to health

OBJECTIVE: To examine the effect of vitamin B12 deficiency on older veterans and its relationship to general health and cognitive impairment. DESIGN: Cross-sectional study. SETTING: Oklahoma City Veterans Affairs Medical Center. PARTICIPANTS: Data for this research were obtained from 303 ambulatory, older veterans who used the outpatient laboratories of the Oklahoma City Department of Veterans Affairs Medical Center. Subjects were included in the study if they were 65 years of age and older and if they had no known diagnosis associated with B12 deficiency. The sample in this study consisted of 301 men and 2 women aged 65 to 89 years. MEASUREMENTS: This study used two separate measurements of vitamin B12 deficiency: (1) a strict definition of B12 deficiency (serum B12 level < laboratory norm) and (2) a broader definition of B12 deficiency (serum B12 level < laboratory norm or laboratory norm < B12 < 300 pg/mL and methyl malonic acid (MMA) or homocysteine (HC) elevated by more than two standard deviations). The laboratory norm is 200 pg/mL. The dependent variables were measures of cognitive impairment and general health. Cognitive impairment was measured using the Folstein Mini-Mental State Examination (MMSE) and general health was measured using the RAND 36-Item Health Survey Version 1.0. The control variables for this study were the subjects' daily alcohol intake, daily intake of a vitamin/mineral supplement, annual income, and level of education. RESULTS/CONCLUSIONS: Nineteen subjects (6%) were vitamin B12-deficient as measured by the strict definition of B12 deficiency (serum B12 level < laboratory norm), and 49 subjects (16%) were vitamin B12-deficient as measured by the broader definition of B12 deficiency (serum B12 level < laboratory norm or laboratory norm < B12 < 300 pg/mL and MMA or HC elevated by more than two standard deviations). Vitamin B12 level decreases as age increases. Of the nine general health outcomes measured by using the RAND 36-Item Health Survey, only bodily pain is associated with vitamin B12 deficiency, and only then when B12 deficiency is measured as serum B12 level < laboratory norm, the strict definition of B12 deficiency. Vitamin B12-deficient subjects experience more bodily pain than those with normal vitamin B12 levels. There is a significant difference between B12-deficient subjects and B12 normal subjects on cognitive impairment, with B12 normal subjects indicating less cognitive impairment, only when B12 deficiency is measured as B12 level < laboratory norm, the strict definition of B12 deficiency. The broader measurement of vitamin B12 deficiency (i.e., serum B12 level < laboratory norm or laboratory norm < B12 < 300 pg/mL and MMA or HC elevated by more than two standard deviations) is not a significant correlate of cognitive impairment and general health.     

Fatty acid composition of myelin isolated from the brain of a patient with cellular deficiency of co-enzyme forms of vitamin B12

Myelin fractionation and subsequent lipid isolation have been carried out on a brain from a patient who suffered from a cellular deficiency of the adenosylcobalamin and methylcobalamin co-enzyme forms of vitamin B12. Examination of the fatty acid composition of choline and ethanolamine glycerophospholipids indicated a relative enrichment of odd-chain fatty acids which were identified by gas-liquid chromatography-mass spectroscopy as C15, C15:1, C17 and C17:1. A mixture of methyl branched C17 fatty acids was also identified. Odd-chain fatty acids accounted for 9.8% of the total fatty acid in the myelin choline phospholipid conpared to control values of 1.2%. The affected brain myelin phospholipids had a lower unsaturated fatty acid content. Examination of the myelin sphingolipids, sphingomyelin, cerebroside and sulfatide, yielded abnormal fatty acid profiles. The sphingomyelin contained only small amounts of C24:1 fatty acid. Both normal and hydroxy fatty acid containing cerebroside and sulfatide had reduced levels of C24 fatty acid. Determination of the relative hydroxy and normal fatty acid content of the galactolipids indicated an abnormally high hydroxy fatty acid level. Abnormal fatty acid profiles of brain cerebral sphingolipids have not been previously described in cases of vitamin B12 deficiency. Whether or not these alterations are characteristic will only be established by estimating sphingolipids in other such cases.     

Ischemic strokes secondary to vitamin B12 deficiency-induced hyperhomocystinemia

A 45-year-old woman sustained two ischemic cerebral infarctions 16 years after ileal resection for Crohn's disease. Her evaluation showed an elevated random serum homocystine level, a low serum vitamin B12 level, and an increased mean corpuscular volume (MCV) without anemia. A methionine-loading test resulted in a marked increase in the homocystine levels 2, 4, and 6 hours after the load. A Schilling test demonstrated a malabsorption of vitamin B12. Vitamin B12 injections normalized her fasting homocystine level and her MCV. She has had no recurrent strokes during a year follow-up.     

Inhibitory effect of eggs on vitamin B12 absorption: description of a simple ovalbumin 57Co-vitamin B12 absorption test

Ovalbumin and egg yolks, mixed separately in vitro with radiocyanocobalamin (57 Co-vitamin B12), were served to normal volunteers in a cooked form. Ovalbumin, and to a lesser degree, egg yolks were observed to inhibit vitamin B12 absorption. This observation explains the rather poor assimilation of vitamin B12 from eggs labelled in vivo with 57 Co-vitamin B12.     

The effect of vitamin B12 on the tolerance of chicks for high levels of dietary fat and carbohydrate

Three experiments were conducted with White Leghorn chicks hatched from hens fed diets varying in levels of protein, fat, and vitamin B12. Adding animal fat at a level of 10% in the chick diet caused growth depression of vitamin B12 deficient chicks, regardless of protein or energy level of hen or chick diet. Increasing the level of fat to 20% in the chick diet caused further growth depression and increased mortality. Feed efficiency of vitamin B12 deficient chicks was severely depressed by each additional increment in the fat level. Increasing protein content from 20 to 30% in the chick diet resulted in severe growth depression and poor feed efficiency. Although the added fat in the 30% protein chick diet depressed growth of chicks hatched from hens fed the 16 and 32% protein with added fat, it improved growth of those hatched from hens fed the similar diets with no added fat. Added fat in the 30% protein chick diet also improved feed efficiency of all chicks regardless of breeder diet treatments. Chicks hatched with an adequate carry-over of vitamin B12 from hens or chicks fed a diet with 10 micrograms of added vitamin B12/kg of feed did not show the growth depression caused by the high level of fat in the 20 and 30% protein chick diets. Feed efficiency was greatly improved by the addition of vitamin B12 to all chick diets. In a 22% protein vitamin B12 deficient diet, isocaloric substitution of glucose for fat depressed chick growth significantly and this growth depression was counteracted by supplementing the diet with 10 or 100 micrograms of vitamin B12/kg of feed. The vitamin B12 requirement was not increased by such substitution in the 22% protein diet. In contrast, isocaloric substitution of fat for glucose in the 32% protein chick diet increased the vitamin B12 need for optimum growth.     

Central administration of vitamin B12 aggravates cataplexy in canine narcolepsy

Experimental evidence in canine narcolepsy suggests that central cholinergic systems are critically involved in the regulation of cataplexy, an abnormal manifestation of REM sleep atonia. In the current study, we found that intracerebroventricular perfusion of methyl-B12, (10(-5)-10(-2) M), significantly aggravated cataplexy and enhanced REM sleep in narcoleptic dogs. Choline, a direct precursor of acetylcholine, was also found to aggravate cataplexy, while cyano-B12, a vitamin B12 analog without methyl donating abilities, had no effect on cataplexy. Since both methyl-B12 and choline are reported to enhance acetylcholine synthesis, enhancement of the biosynthesis of acetylcholine may be involved in the effects observed in canine narcolepsy. Our results suggest that central administration of methyl-B12 has the potential to modulate both normal and pathological REM sleep.     

Megaloblastic anaemia in childhood due to vitamin B12 deficiency, report of 3 cases of congenital selective vitamin B12 malabsorption (author''s tranls)

Three cases of congenital selective malabsorption of vitamin B12 (Imerslund-Grasbeck syndrome) are presented. Pathophysiological aspected and clinical symptoms of this disease are discussed together with other megaloblastic anaemias in childhood caused by vitamin B12 deficiency.